Abstracts

Rationale: Neuronal chloride concentration ([Cl-]i) is an important determinant of both post-synaptic GABAA-receptor mediated signaling and cell volume regulation. Altered Cl- equilibrium results in cell swelling, acute and chronic accumulation of [Cl-]i, and GABA depolarizing responses, which foster seizures, epileptogenesis and anticonvulsant resistance via failure of inhibition. Cl- equilibrium is mediated by the cation-chloride co-transporters (CCCs) KCC2 (a canonical Cl- exporter) and NKCC1 (a canonical Cl- importer). Pathological alterations in CCC activities may contribute to Cl- disequilibrium after brain injury. Our goal was to test whether antagonizing NKCC1 activity and / or stimulating KCC2 activity comprised a useful therapeutic strategy to reduce cell swelling and [Cl-]i in injured neurons, restore GABAergic inhibition, suppress seizures, and prevent epileptogenesis. Bumetanide and furosemide are well-known inhibitors of CCCs. New drugs that modulate CCCs activity include CLP257 that selectively activates KCC2, and the VU series (0240551, 0255011, 0726722) that selectively inhibit KCC2.Methods: We tested the mechanism of action and specificity of the putative KCC2 agonist CLP257 and KCC2 inhibitor D4.2 (VU0726722) in the organotypic hippocampal slice in vitro model of post-traumatic epileptogenesis. Extracellular field potential recordings, two-photon imaging of the transgenic chloride fluorophore Clomeleon, and lactate and lactate dehydrogenase (LDH) production assays were used to monitor neuronal network activity, [Cl-]i and neuronal cell death.Results: We found that: (i) CLP257 reduced the amount of early and late chronic seizure activity in a concentration-dependent manner, assayed by lactate and LDH production; (ii) the GABAA–R antagonist SR95531 (10 μM) as well as the NKCC1 and KCC2 blocker bumetanide (10 and 200 μM) prevented the acute antiepileptic action of CLP257 (30 μM); (iii) The VU KCC2 antagonists (10 μM) strongly increased the amount of early chronic seizure activity and the rate of neuronal death, assayed by lactate and LDH production; (iv) VU (10 μM) pro-convulsive action was not affected by block of GABAA-R and CCCs.Conclusions: Our results indicate that: 1) the antiepileptic action of CLP257 more likely was mediated by suppression of NKCC1 rather than by activation of KCC2; 2) the pro-epileptic action of VU was not mediated by CCC modulation. Our data validate CLP257 as a promising target of investigation for antiepileptic therapies, and highlight the ongoing need to develop more specific activators and inhibitors of KCC2 co-transport.